The present invention relates to a method for engrafting human hematopoietic cells in a non-human mammal by bone marrow transplantation. More particularly, the present invention relates to chimeric mammals with human hematopoietic cells, including progenitor cells and mature cells of the myeloid, lymphoid and erythroid lineages, a process for engrafting human hematopoietic cells to create a chimeric mammal, and a process for creating hybridoma cells capable of producing a human monoclonal antibody.
The mammalian hematopoietic system is arranged as a hierarchy consisting of a wide array of cells ranging from large numbers of mature differentiated cells to rare pluripotent stem cells capable of self-renewal and differentiation. Much of our knowledge concerning organization and regulation of mammalian hematopoietic systems is derived from studies with murine hematopoietic systems. Conversely, our understanding of specifics of the human hematopoietic system is much less complete due to a lack of a reliable model for human hematopoiesis. Therefore, there is a need in the art for a human-specific hematopoiesis model that includes human stem cells.
Various investigators have attempted to transplant human hematopoietic cells into a murine bone marrow microenvironment with varying degrees of success. Xenogeneic bone marrow transplantation is possible between closely related species, such as rat and mouse. However, xenogeneic bone marrow transplantation has not enabled the stable transplant of human hematopoietic cells of multiple lineages into more distantly related mammalian species, such as mice or rats. For example, engraftment of human cells in severe combined immune deficiency (SCID) mice, permitted infection of mice with human immunodeficiency virus (HIV) (Namikawa et al., J. Exp. Med. 172:1055, 1990). However, detectable human cells were limited to a human T cell lineage and survived for only two to three months. Further, the T cells were detected only in blood and not in any other organ. Thus, transplantation of human hematopoietic cells into SCID mice was limited to the transient survival of only one lineage of human hematopoietic cells.
McCune et al. (Science 241:1632, 1988) attempted to facilitate engraftment of human hematopoietic cells (from human fetal liver) into a SCID mouse by further transplanting a graft of human fetal thymic epithelium under the kidney capsule. Control SCID mice that were tansplanted with human hematopoietic cells but not fetal thymic epithelium did not engraft or differentiate into human T cells. No other human cell lineage except human T cells were detected in the mouse tissues. Thus, it appears that transplanted human hematopoiesis observed by McCune et al. occurred only in implanted human thymic tissue and that no engraftment of human cells in murine tissue actually occurred.
EP-A438053 describes an attempt to create a mouse-human chimeric animal. Bone marrow cells from SCID mice and human bone marrow cells were added to another strain of mouse that was lethally irradiated. SCID mouse bone marrow was used because T and B lymphocytic lineages in SCID mice cannot develop from SCID pluripotent stem cells due to a deficiency in rearrangement of antigen receptor genes (Shuler et al., Cell 46:963, 1986). The resulting chimeric mice had a small number of human cells in the thymus with a predominant CD3+ CD4+ CD8+ phenotype. Lubin et al. Science 252:427, 1991 cotransplanted a T cell depleted human bone marrow with SCID bone marrow into lethally irradiated Balb/c mice. Lubin et al. found only T cells and B cells in blood over several months. Therefore, this procedure did not produce multilineage engraftment of human hematopoietic cells.
Kamel-Reid et al. (Science 242:1706, 1988) report the engraftment of human bone marrow cells into SCID mice without implantation of fetal tissue. However, observation periods were limited to thirty days, so long term engraftment was not determined.
Therefore, recent progress to establish a stable chimeric animal model to characterize normal development of human hematopoietic stem cells has been limited to transient or incidental engraftment of one or two human lineages. A chimeric mouse with a high proportion (i.e., predominance) of human hematopoietic cells has not yet been achieved. Such a chimeric mammal would be useful as an experimental model for studying a variety of human and veterinary diseases. Such a chimeric mammal would also be useful for studying normal human hematopoiesis, human stem cells and all of their progeny and those growth and inhibitory factors that govern hematopoiesis. Further, a chimeric mammal with functioning human immune cells would be useful to produce human monoclonal antibodies. The present invention was made to improve techniques used to produce chimeric mammals with human hematopoietic cells.
The present invention provides a chimeric mammal having a stable bone marrow graft of human hematopoietic cells capable of differentiating into multiple lineages of mature human cells, wherein at least 30% of the hematopoietic cells in the mammal""s bone marrow are of human origin. The chimeric mammal is preferably a mouse, and most preferably a SCID mouse. Further, the graft consists essentially of human hematopoietic progenitor cells, multipotent human progenitor cells and mature human hematopoietic cells of lymphoid, myeloid and erythroid lineages.
The inventive method comprises sublethally irradiating an immunodeficient mammal, infusing human hematopoietic cells into the mammal and administering an effective amount of human mast cell growth factor (MGF) and a human granulocyte macrophage colony stimulating factor/interleukin-3 fusion protein (GM-CSF/IL-3 FP) to promote engraftment of human hematopoietic cells. Preferably, GM-CSF/IL-3 FP is a specific fusion protein called PIXY321.
The present invention further provides a chimeric mammal having a stable bone marrow graft of lineage-specific human hematopoietic cells, wherein the lineage-specific human hematopoietic cells are selected from the group consisting of erythroid cells, myeloid cells, lymphoid cells, multipotent progenitor cells and combinations thereof. A process for obtaining a chimeric mammal having a stable bone marrow graft of lineage-specific human hematopoietic cells comprises, sublethally irradiating an immunodeficient mammal, infusing human hematopoietic cells into the immunodeficient mammal, and administering MGF, a GM-CSF/IL-3 FP and an additional, lineage-specific human growth factor which is effective to promote differentiation and proliferation of the desired lineage of human hematopoietic cells. Additional, lineage-specific human growth factors are selected from the group consisting of erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), interleukins-2, -4, -5, -6, -7, -9, -10, -11, and -12, and combinations thereof.
Further still, the present invention provides a human hybridoma cell capable of producing a human monoclonal antibody (hMAb) specific for a selected antigen. The human hybridoma cell is made by a process comprising immunizing a chimeric mammal made according to the inventive process, fusing spleen cells from the chimeric mammal with myeloma cells to make hybridoma cells, and selecting for hybridoma cells which secrete hMAbs that bind to the antigen.
The present invention involves various methods of using MGF and a GM-CSF/IL3 FP, such as PIXY321, to promote engraftment of transplanted human hematopoietic cells into various mammalian species. We have significantly improved upon previous methods to create a human-chimeric mammal having at least 30% human cells in the chimeric mammal""s bone marrow. Quite often, the proportion of human origin cells versus host mammal hematopoietic cells in the chimeric mammal bone marrow often exceeds 40% or 50% human cells. This level (i.e., at least 30%) of human origin cells of multiple lineages has never been achieved before the present invention. Further, such human cells are capable of differentiating into multiple lineages of mature human cells, such as myeloid, lymphoid and erythroid lineages.
The inventive method involves sublethally irradiating an immunodeficient mammal, such as a SCID mouse, transplanting immature human hematopoietic cells (i.e., from bone marrow, cord blood or peripheral blood), and administering a human engraftment growth factor composition, comprising at least MGF and and a GM-CSF/IL-3 FP, such as PIXY321. The improvement in the inventive method that achieves stable and long-term transplantation of multiple lineages of human hematopoietic cells is administration of an engraftment growth factor composition comprising human-specific cytokines. Preferably, the engraftment growth factor composition is administered by injection (i.e., intraperitoneal, intravenous, subcutaneous, etc) and at least thrice weekly for at least two weeks.
Most preferably, the engraftment composition further comprises an additional, lineage-specific, human cytokine to stimulate proliferation of one or more specific lineages of human hematopoietic cells, wherein the additional, lineage-specific, human cytokine is selected from the group consisting of interleukin-7 (IL-7), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), interleukin-1 (IL-1xcex1 or IL-1xcex2), interleukin-2 (IL-2), erythropoietin (EPO), interleukin-5 (IL-5), interleukin-4 (IL-4), interleukin-6 (IL-6), and combinations thereof. The additional human cytokine promotes proliferation of more mature or differentiated human myeloid, erythroid, or lymphoid lineage hematopoietic cells.
The present invention further provides a method for preparing a human monoclonal antibody (hMAb) in the inventive chimeric mammal. The hMAb is prepared by first immunizing a chimeric mammal with a selected antigen. Spleen cells from the immunized chimeric mammal are collected and fused with myeloma cells to form hybridoma cells. Human MAbs from supernatants of hybridoma cell cultures are assayed to select hMABs that bind to the selected antigen.
Further still, the present invention provides a chimeric mammal capable of providing a renewable supply of mature human blood cells, such as erythrocytes, monocytes, granulocytes or lymphocytes. The chimeric mammal is made according to the present invention utilizing a human growth factor composition supplemented with a lineage-specific human growth factor. The addition of the lineage-specific growth factor will selectively augment proliferation of a specific lineage of human cell to promote proliferation and differentiation of mature human cells of the specific lineage. Examples of lineage-specific human growth factors include EPO for erythrocytes, G-CSF for neutrophils, IL-2, IL-7, IL-9 or IL-10 for T cells, IL4, IL-6 or IL-7 for B cells, IL-5 for eosinophils and GM-CSF or M-CSF for monocytes.
Chimeric mammals, according to the present invention, are useful for models of human disease, particularly, thalassemia where such chimeric mammals (transplanted with human bone marrow cells from a patient with such a disease) would be useful for preclinical assessment of various treatments. Moreover, chimeric mammals (particularly rats or mice) with a high percentage of human cells in bone marrow (i.e., at least 30%) would be further useful to support human tumor cell growth and serve as a specific model for human leukemia. Lastly larger chimeric mammals (e.g., sheep) can be used as a source of lineage specific cells, such as human erythrocytes when an additional, lineage-specific growth factor is used.
In a preferred method for engrafting human hematopoietic progenitor cells into a living mammal, the inventive process comprises first irradiating an immunodeficient mammal with a sub-lethal dose of irradiation. An immunodeficient mammal, as defined herein, is a mammal that lacks a significant population of functioning, host-specific, T cells and B cells. One example of an immunodeficient mammal is a SCID mouse. A SCID mouse is described, for example, in Kamel-Reid et al., supra. Other examples of immunodeficient mammals include nude, beige, xid (X-linked immune deficiency), and nod (non-obese diabetic) mice. A sub-lethal dose of irradiation is, for example, from about 1000 to about 100,000 rads of radiation per kilogram body weight of the immunodeficient mammal.
The next step involves infusing from about 106 to about 109 human hematopoietic cells per kilogram body weight to the immunodeficient, irradiated mammal. Human hematopoietic cells can be obtained from human bone marrow or human peripheral blood progenitor cells by standard techniques. Human hematopoietic cells (i.e., mononuclear cells) can be separated from bone marrow or peripheral blood, for example, by density gradient sedimentation (e.g., Ficoll).
Engraftment of the human hematopoietic cells from bone marrow or peripheral blood is promoted by administering an effective amount of an engraftment growth factor composition wherein said engraftment growth factor composition comprises hMGF and GM-CSF/IL-3 FP. Preferably, the GM-CSF/IL3 FP is PIXY321. The engraftment growth factor composition is administered at least thrice weekly (by intravenous, intraperitoneal, subcutaneous, intramuscular or other means of systemic administration) for at least two weeks. The engraftment growth factor composition should be administered within one month after administering human hematopoietic cells. Preferably, the engraftment growth factor composition is administered daily for at least one month immediately following human hematopoietic progenitor cell administration. Every other day or thrice weekly administration of the engraftment growth factor composition can also be effective. Most preferably, administration of the engraftment growth factor composition begins immediately after transplantation with human bone marrow.
Mast cell growth factor (MGF) has also been called Steel factor (SF), stem cell factor (SCF), and Kit Ligand (KL). All of the names for this factor refer to the ligand for the c kit proto-oncogene. MGF has been described in a series of seven papers in the Oct. 5, 1990 issue of Cell (Williams et al., Cell 63:167, 1990; Copeland et al., Cell 63:175, 1990; Flanagan and Leder, Cell 63:185, 1990; Zsebo et al., Cell 63:195, 1990; Martin et al., Cell 63:203, 1990; Zsebo et al., Cell 63:213, 1990; Huang et al., Cell 63:225, 1990; and Anderson et al., Cell 63:235, 1990). Expression of various recombinant forms of MGF has been described in U.S. patent application Ser. No. 07/586,073, filed Sep. 21, 1990 and U.S. patent application Ser. No. 07/713,715 filed Jun. 12, 1991, the disclosures of which are incorporated by reference herein. MGF has been found to stimulate proliferation and recruitment of early myeloid and lymphoid lineage progenitor cells and possibly even the most primitive hematopoietic stem cells. Effective amounts of hMGF are from about 100 to about 10,000 xcexcg per kilogram per day.
Fusion proteins comprising GM-CSF and IL-3 components are described in U.S. patent application Ser. No. 07/567,983 filed Oct. 14, 1990, the disclosure of which is incorporated by reference herein. A particular GM-CSF/IL-3 fusion protein (PIXY321) has been found to interact with GM-CSF receptors and/or IL-3 receptors (Curtis et al., Proc. Natl. Acad. Sci. USA 88:5809, 1991). PIXY321 is a GM-CSF/IL-3 fusion protein having Leu23 Asp27 Glu34 hGM-CSF/Gly4 Ser Gly5 Ser/Pro8 Asp15 Asp70 hIL-3. Thus, this protein comprises a triply-substituted GM-CSF domain fused to a doubly-substituted Pro8 IL-3 domain via a linker or spacer domain comprising glycine and serine residues. A preferred dose of GM-CSF/IL3 FP or PIXY321 is from about 25 to about 2,500 xcexcg per kilogram per day.
Additional, lineage-specific human cytokines are known in the art and described in various publications. More specifically, interleukin-1 has been found to exist in two forms, IL-1xcex1 and IL-1xcex2 (March et al., Nature 315:641, 1985). Both IL-1xcex1 and IL1xcex2 bind to IL-1 receptors (Type I and Type II) to transduce signal. Il-1xcex1 is active in both precursor and mature forms, whereas IL-1xcex2 is only active in mature form but not in precursor form (March et al., supra). Il-1 also includes active fragments and analogs with altered amino acid sequences and derivatives, such as fusion proteins having an IL-1 component and IL-1 biological activity (Mosley et al., Proc. Natl. Acad. Sci. USA 84:4572, 1987). An appropriate dose for IL-1 is from 50 to 5,000 ng/kg/day.
Interleukin-2 has been described in U.S. Pat. No. 4,738,927, EPO has been described in U.S. Pat. No. 4,703,008, interleukin-7 has been described in U.S. Pat. No. 4,965,195, interleukin-4 has been described in U.S. Pat. No. 5,017,691, interleukins-5 and -6 have been described in Campbell et al., Proc. Natl. Acad. Sci. USA 84:6629, 1987 and Chiu et al., Proc. Natl. Acad. Sci. USA 85:7099, 1988, respectively. Interleukins-9 and -10 have been described in Yang et al., Blood 74:1880, 1989 and Vieira et al., Proc. Natl. Acad. Sci. USA 88:1172, 1991, respectively. Interleukins -11 and -12 have been described in Paul et al., Proc. Natl. Acad. Sci. USA 87:7512, 1990 and Gubler et al., Proc. Natl. Acad. Sci. USA 88:4143, 1991, respectively. Appropriate doses for interleukins-2, -4, -5, -6, -7, -9, -10, -11 and -12 are from 50 to 10,000 ng/kg/day. An appropriate dose range for EPO is from 10 to 1,000 U/kg/day. G-CSF and M-CSF are described in U.S. Pat. No. 4,810,643 and Wong et al., Science 235:1504, 1987, respectively, and an appropriate dose for both is from about 50 to about 10,000 vg/kg/day.
A preferred method for making a hybridoma cell line that secretes a human monoclonal antibody is to inject a chimeric mammal, according to the present invention, with a selected antigen. Preferably, the chimeric mammal is a mouse and most preferably a SCID mouse. Preferably, there are four intraperitoneal injections, approximately one week apart, with about 10 xcexcg of antigen per injection in an adjuvant, preferably Freund""s adjuvant. After inoculation the mammals are tested for antibody levels in a test bleeding. Antibody secreting mammals have their spleens removed. Spleen cells are fused with myeloma cells. Suitable myeloma cells include, for example, AG8.653, NS1, and NS0. Hybridoma cells are formed with this fusion.
The hybridoma cells secrete monoclonal antibodies into a culture supernatant. Supernatants are screened for human monoclonal antibodies (hMAb) that both bind to the antigen and are human antibodies. Such screening can be performed in one step. For example, a 96 well plate can be coated with an anti-human rabbit immunoglobulin, and supernatant from the hybridoma cells and labelled antigen added. After washing, only antibodies that are both human and bind to the antigen will form a detectable sandwich within the well. Such hMAbs are selected from this detection.
The present invention is directed to a chimeric mammal. The chimeric mammal is created by employing intravenous (iv) injection of adult human bone marrow into immunodeficient mice conditioned with radiation. Conditioning helps to suppress rejection mechanisms and to promote stem cell engraftment. In earlier attempts to create a chimeric mouse, human macropage cells and their macrophage precursors accumulated in murine bone marrow, increased in number (although nowhere near the percentage of human cells detected according to the present invention), and were maintained in this environment for several months, (Kamel-Reid et al., supra). However, no mature cells were detected (Id.). This procedure was refined in Lubin et al., supra. Therefore, the present invention describes an improved procedure to promote engraftment of human hematopoietic cells of multiple lineages, including granulocytes, macrophages, B cells, erythroid, and multilineage progenitor cells into a non-human mammal (e.g., murine) microenvironment. This supports long term proliferation and differentiation of human hematopoietic stem cells and pluripotent precursor cells of many lineages.
We conducted experiments with sub-lethally irradiated SCID mice transplanted with human hematopoietic cells, found that administration of human engraftment growth factors MGF and PIXY321 (a GM-CSF/IL-3 FP) promotes repopulation of murine bone marrow with human hematopoietic progenitor cells and human mature cells of myeloid, lymphoid and erythroid lineages. When EPO was administered as an additional, lineage-specific human growth factor, mature human red blood cells (HRBC) appeared in the blood of the mice after 30 days of human engraftment growth factor and additional, lineage-specific growth factor administration.
In a group of experiments, SCID mice were sub-lethally irradiated with, for example, 400 cGy, 137Cs. A lethal dose for a relatively small mouse, such as a SCID mouse, would be at least 500 cGy. Human bone marrow cells from normal volunteers were purified over a density gradient (e.g., Ficoll, Pharmacia) and injected into the sublethally irradiated SCID mice by tail vein injection (i.v.). The mice were administered (i.p.) a human engraftment growth factors composition (PIXY321 @ 8 xcexcg/mouse/2days and MGF @ 20 xcexcg/mouse/2days) every other day for at least two weeks.
The relative amount of human cells in bone marrow of chimeric mammals was determined by sacrificing the mammal (21 to 40 days after human cell infusion), extracting DNA from bone marrow or any other tissue of interest (such as spleen) and analyzing for the presence of human DNA by established procedures. One such procedure for analyzing for human DNA is described in Kamel-Reid et al. Science 242:1706, 1988 or Kamel-Reid et al., Science 246:1597, 1989. Briefly, DNA was extracted from bone marrow, digested with Eco R1, blotted according to standard procedures, and probed with p17H8 (described in Wave et al., Nucleic Acids Res. 15:7549, 1987). p17H8 is a human xcex1-satellite probe specific for sequences on human chromosome 17. Eco R1 digestion of human DNA produces a characteristic 2.7 kb band from human chromosome 17 visible by autoradiography. Autoradiograms were analyzed with a video densitometer (BioRad, Richmond, Calif.) to quantitate 2.7 kb band intensity. Percentage of human cells was determined by comparison to standards with a known percentage of human DNA.
Transplanting human hematopoietic cells into sub-lethally irradiated SCID mice, without using any growth factors, obtained, at most, up to 2 or 3% human cells in bone marrow. Engraftment growth factor addition dramatically increased the proportion of human cells populating chimeric SCID mouse bone marrow to at least 30% human cells. In a comparative experiment the proportion of SCID mice (sub-lethally irradiated and administered 4xc3x97107 human bone marrow mononuclear cells) administered a human engraftment growth factor composition, showed at least a ten fold increase in the level of human cells engrafting in murine bone marrow over those chimeric mice not treated with growth factors. In some mice, the stimulatory effect of a human engraftment growth factor composition exceeded 100-fold. In each experiment, mice receiving human engraftment growth factors, according to the present invention, exhibited levels of human DNA in bone marrow exceeding ten fold the level of human DNA measured in control mice that were not administered human engraftment growth factors. All experiments measured percentage of human cells in bone marrow by determining the intensity of the characteristic 2.7 kb human chromosome 17 xcex1-satellite band in Eco R1-digested DNA. Human/mouse DNA mixtures of known proportions were used as standards.
We determined whether or not human stem cells had engrafted into SCID mice using the inventive procedure. Human stem cells can only be identified indirectly by their function, such as the ability to differentiate into all cell lineages, high degree of self-renewal capacity, slow cell cycling and an ability to engraft for long periods of time. We determined that true human stem cells were able to engraft in a murine bone marrow in chimeric SCID mice produced according to the inventive procedure.
By comparison, when human PBL (peripheral blood lymphocytes, a more mature cell line) were used for transplantation of human cells (instead of human hematopoietic cells from bone marrow or peripheral blood) no human cells were detected in bone marrow of such mice. Therefore, transplantation of mature or more differentiated human lymphoid cells was not responsible for characteristic multilineage engraftment seen in the inventive chimeric mammals. Secondly, continued presence of myeloid and erythroid progenitor cells, and multipotent progenitor cells in the inventive chimeric mammals suggests that earlier precursor cells were able to maintain a pool of human progenitor cells of all lineages. Moreover, we found CD34+ cells in chimeric SCID mice administered an engraftment growth factor composition. This is consistent with a notion that a pool of early progenitor cells had engrafted in bone marrow of inventive chimeric mammals.
In another experiment, SCID mice were transplanted with human bone marrow cells and administration of an engraftment growth factor composition was delayed for one month following transplantation. These mice contained low levels of human myeloid progenitor cells, no detectable erythroid or multilineage progenitor cells, and no detectable mature cells of any lineage at the time the engraftment growth factor composition was administered. One to three months after transplantation, the chimeric mice were administered an engraftment growth factor composition comprising PIXY321 and MGF and, in some instances, a lineage-specific growth factor EPO in addition to the engraftment growth factor composition. High levels of human cells of all lineages were observed in mice receiving the engraftment growth factor composition with or without EPO. Only those mice administered EPO showed detectable BFU-E activity and had significant numbers of CD19+ cells. These data provide evidence that human pluripotent stem cells had engrafted in mouse bone marrow and were responding to human growth factors. These data further suggest that it is possible to select for specific lineages of human hematopoietic cells by administering an additional, lineage-specific human growth factor, such as EPO for erythroid cells.